The baculovirus expression vector system (BEVS) is a proven, powerful and versatile method of eukaryotic protein expression. It is used to produce vaccines, diagnostics, and biologically active proteins for a multitude of research projects. Like all expression systems, however, BEVS has its disadvantages. One is the fact that expression is short-lived due to virus-induced cell death and lysis. ParaTechs has already commercialized a product that addresses this shortcoming. Cell lines that express a viral ankyrin gene show delayed death and lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production. This activity, referred to as vankyrin-enhanced BEVS (VE-BEVSTM), boosts target protein expression up to 22-fold. A second limitation of baculovirus expression is that insect cells lack the ability to produce terminally sialylated, complex N-glycans, which limits the usefulness of BEVS for the expression of human therapeutic proteins. GlycoBac LLC has developed a transgenic insect cell line (SfSWT4) that expresses six mammalian glycosylation enzymes, allowing synthesis of terminally sialyated proteins. This Phase II proposal combines ParaTechs' VE technology with GlycoBac's cell line to optimize expression of humanized N-glycans. In Phase I, the transgenic cell line SfSWT4 was transformed with several vankyrin genes under the control of different promoters. Polyclonal cells were screened for enhanced glycoprotein expression. Data demonstrating that infected cells lived longer and produced more authentically sialylated protein confirmed our hypothesis. Phase II will extend these studies by (1) cloning and characterization of VE-SWTTM cells according to FDA guidelines for cells used to produce vaccines and biological; (2) examining synergistic effects between VE virus vectors and VE-SWT cell lines to provide greater levels of enhancement; and (3) demonstrating enhanced expression of medically relevant glycoproteins in VE-SWTTM cells. These Phase II studies will significantly expand the applications of ParaTechs' VE-BEVS technology and GlycoBac's glycoengineered cell lines. Personnel at both companies have experience with the techniques to be used. Preliminary studies indicate that this technology has a significant chance of performing as envisioned. Furthermore, prior marketing experience with transformed cell lines previously released from the two companies suggests a significant demand for the expanded technology. This new technology should be relatively easy to commercialize based on the established reputations of ParaTechs, Inc. and GlycoBac, LLC and the growing demand for improved cell lines to express recombinant humanized glycoproteins. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of the baculovirus expression system for the production of human therapeutic proteins. This proposal addresses that deficiency and aims to develop new cell lines that produce authentic humanized N-glycans in the context of ParaTechs' vankyrin-enhanced baculovirus technology. Successful completion of these objectives will produce cells that provide the highest levels of accurately processed secreted and transmembrane proteins and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics.